This invention relates to the crystallization and structure determination of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (S. aureus MurB).
Reports of an increase in antibiotic resistant bacteria have stimulated efforts to find new classes of therapeutic agents that will prevent society from entering a xe2x80x9cpost-antibiotic age.xe2x80x9d Historically, three important cellular functions have been the major targets of antibioticsxe2x80x94cell wall biosynthesis, DNA replication, and protein translation. The biosynthesis of the bacterial cell wall, in particular the peptidoglycan polymer, is a particularly attractive target since this flexible structure provides protection for the cell against osmotic lysis. To date, most of the therapeutic agents discovered that target cell wall biosynthesis inhibit the later stages of peptidoglycan biosynthesis at the point where interstrand cross linking occurs between the peptide chains. Recent efforts have been directed toward purifying and characterizing all the enzymes in the peptidoglycan biosynthetic pathway with an eye toward designing novel enzyme inhibitors of these essential targets.
Bacterial peptidoglycan is a polymer which includes a repeating disaccharide subunit of N-acetylglucosamine and N-acetylmuramic acid and an extended four to five residue amino acid chain. The first step toward creating this peptidoglycan polymer involves the formation of UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine by the enzymes MurA and MurB. MurA catalyzes the first stage of this transformation by transferring the enolpyruvate moiety of phosphoenolpyruvate to the 3xe2x80x2 hydroxyl of UDP-N-acetylglucosamine with the release of inorganic phosphate. The resulting product, enolpyruvyl-UDP-N-acetylglucosamine (EP-UDPGlcNAc), undergoes a reduction catalyzed by the MurB enzyme by utilizing one equivalent of NADPH and a solvent derived proton. This two electron reduction creates the lactyl ether of UDP-N-acetylmuramic acid upon which a five residue peptide chain is built. Construction of this pentapeptide is catalyzed in a nonribosomal fashion by the enzymes MurC, MurD, MurE, and MurF (FIG. 1) in both Gram negative bacteria such as Escherichia coli and Gram positive bacteria such as Staphylococcus aureus. The resulting UDP-N-acetylmuramyl pentapeptide is subsequently attached to an undecaprenyl lipid moiety by MraY and joined to another sugar, UDP-N-acetylglucosamine by MurG. In Staphylococci the next steps of peptidoglycan biosynthesis involve another family of enzymes, FemX, FemA, and FemB which create a pentaglycine strand in a stepwise fashion on the amino terminus of the lysine side chain. This extended Lys-Gly5 chain serves as the interstrand bridge between nearby peptide strands. Crosslinking between strands can then occur between the lysine-pentapeptide bridge and the carbonyl of the fourth residue (D-Ala) with release of the terminal D-Ala in a transpeptidation step catalyzed by penicillin binding proteins.
While several laboratories have characterized some of the peptidoglycan biosynthetic enzymes for E. coli little biochemistry or structural biology has been carried out on these enzymes in a clinically relevant Gram positive organism. Interest in the molecular mechanisms of peptidoglycan biosynthesis in Gram positive organisms has increased in recent years as methicillin resistant S. aureus strains have surfaced that have acquired resistance to the antibiotic vancomycin.
In one aspect, the present invention provides a method for crystallizing an S. aureus MurB molecule or molecular complex that includes preparing purified S. aureus MurB at a concentration of about 1 mg/ml to about 50 mg/ml and crystallizing S. aureus MurB from a solution comprising about 1 wt. % to about 50 wt. % PEG, 0 wt. % to about 40 wt. % DMSO, about 100 mM to about 1 M ammonium or lithium sulfate, about 0 mM to about 20 mM 2-mercaptoethanol, about 0.005 mM to about 40 mM EP-UDPGlcNAc substrate, and buffered to a pH of about 5 to about 8.
In another aspect, the present invention provides crystalline forms of an S. aureus MurB molecule. In one embodiment, a crystal of an S. aureus MurB is provided having the trigonal space group symmetry I213.
In another aspect, the present invention provides a scalable three dimensional configuration of points derived from structure coordinates of at least a portion of an S. aureus MurB molecule or molecular complex. In one embodiment, the scalable three dimensional set of points is derived from structure coordinates of at least the backbone atoms of the amino acids representing a FAD and/or substrate binding pocket of an S. aureus MurB molecule or molecular complex. In another embodiment, the scalable three dimensional set of points is derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to an S. aureus MurB molecule or molecular complex. On a molecular scale, the configuration of points derived from a homologous molecule or molecular complex have a root mean square deviation of less than about 1.0 xc3x85 from the structure coordinates of the molecule or complex.
In another aspect, the present invention provides a molecule or molecular complex that includes at least a portion of an S. aureus MurB FAD and/or substrate binding pocket. In one embodiment, the S. aureus MurB FAD binding pocket includes the amino acids listed in Table 1, preferably the amino acids listed in Table 2, and more preferably the amino acids listed in Table 3, the FAD binding pocket being defined by a set of points having a root mean square deviation of less than about 1.7 xc3x85, preferably less than about 1.0 xc3x85, from points representing the backbone atoms of the amino acids. In another embodiment, the S. aureus MurB substrate binding pocket includes the amino acids listed in Table 4, preferably the amino acids listed in Table 5, and more preferably the amino acids listed in Table 6, the substrate binding pocket being defined by a set of points having a root mean square deviation of less than about 1.0 xc3x85 from points representing the backbone atoms of the amino acids.
In another aspect, the present invention provides molecules or molecular complexes that are structurally homologous to an S. aureus MurB molecule or molecular complex.
In another aspect, the present invention provides a machine readable storage medium including the structure coordinates of all or a portion of an S. aureus MurB molecule, molecular complex, a structurally homologous molecule or complex, including structurally equivalent structures, as defined herein, particularly an FAD or substrate binding pocket thereof, or a similarly shaped homologous binding pocket. A storage medium encoded with these data is capable of displaying on a computer screen, or similar viewing device, a three-dimensional graphical representation of a molecule or molecular complex which comprises a binding pocket or a similarly shaped homologous binding pocket.
In another aspect, the present invention provides a method for identifying inhibitors, ligands, and the like of an S. aureus MurB molecule by providing the coordinates of a molecule of S. aureus MurB to a computerized modeling system; identifying chemical entities that are likely to bind to or interfere with the molecule (e.g., screening a small molecule library); and, optionally, procuring or synthesizing and assaying the compounds or analogues derived therefrom for bioactivity. In another aspect, the present invention provides methods for designing inhibitors, ligands, and the like by providing the coordinates of a molecule of S. aureus MurB to a computerized modeling system; designing a chemical entity that is likely to bind to or interfere with the molecule; and, optionally, synthesizing the chemical entity and assaying the chemical entity for bioactivity. In another aspect, the present invention provides inhibitors and ligands designed by the above method. In one embodiment, a composition is provided that includes an inhibitor or ligand designed or identified by the above method. In another embodiment, the composition is a pharmaceutical composition.
In another aspect, the present invention provides a method involving molecular replacement to obtain structural information about a molecule or molecular complex of unknown structure. The method includes crystallizing the molecule or molecular complex, generating an x-ray diffraction pattern from the crystallized molecule or molecular complex, and applying at least a portion of the structure coordinates set forth in FIG. 4 to the x-ray diffraction pattern to generate a three-dimensional electron density map of at least a portion of the molecule or molecular complex.
In another aspect, the present invention provides a method for homology modeling an S. aureus MurB homolog.
Two crystallographic data sets (with structure factors F) are considered isomorphous if, after scaling,             Δ      ⁢              xe2x80x83            ⁢      F        F    =            ∑              "LeftBracketingBar"                              F            1                    -                      F            2                          "RightBracketingBar"                    ∑              F        1            
is less than about 35% for the reflections between 8 xc3x85 and 4 xc3x85.
The following abbreviations are used throughout this disclosure:
UDP-N-acetylenolpyruvylglucosamine reductase (MurB).
Uridine diphospho-N-acetylglucosamine (UDPGlcNAc).
Uridine diphospho-N-acetylglucosamine enolpyruvate (EP-UDPGlcNAc).
Uridine diphospho-N-acetylmuramic acid (UDPMurNAc).
Reduced xcex2-nicotinamide adenine dinucleotide phosphate (NADPH).
Isopropylthio-xcex2-D-galactoside (IPTG).
Dithiothreitol (DTT).
Flavin adenine dinucleotide (FAD).
Dimethyl sulfoxide (DMSO).
Multiple anomalous dispersion (MAD).
The following amino acid abbreviations are used throughout this disclosure: